Treatment of effect of chemicals with their ultradilute stereoisomers

ABSTRACT

A method of treating an effect of a chemical agent, which agent is characterized by one or more chiral centres, by administering a dilution or an ultra-high dilution or potentised preparation of a stereoisomer of said chemical agent.

The present invention relates to a method of treatment, in particular toa method of treating an effect of a chemical agent by administering adilution or an ultra-high dilution or potentised preparation of astereoisomer of the chemical agent.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Homoeopathy employs minute doses of usually harmful or toxic agents tostimulate organisms back to health. The agents used in homoeopathy areselected precisely on the basis of their ability to induce disease-likesymptoms and signs in healthy people when administered in toxic doses,or one or more times in sub-harmful doses. These agents will in properlydiluted form cure a sick person with similar symptoms. While microdoseeffects are now well accepted for example in the phenomenon known ashormesis, some homoeopathic solutions are attenuated beyond Avogadro'sconstant, i.e., in theory none of the original agent remains.Notwithstanding the apparent absurdity of homoeopathic dilutions therehave been a great number of reported experiments which demonstrate thathomoeopathic remedies are effective in treating a variety of symptoms.

Many chemical agents induce undesirable effects on organisms such asmammals. The effects which result from chemical agents having one ormore chiral centres often result from a specific stereoisomer of thechemical agent. For example, (−)-adrenaline is the isomer which is foundin humans and is the chemically active agent. It is about 15 times moreactive than (+)-adrenaline physiologically. Although chemically hard todifferentiate in vitro, in vivo they are readily differentiated by thestereo-specificity of enzymes.

The inventors have now found that the effects of a chemical agent can betreated by administering a dilution or an ultra high dilution orpotentised dilution of a stereoisomer of the chemical agent.Accordingly, there is provided a method of treatment of an organismsuffering from the effects of a chemical agent having one or more chiralcentres, said method comprising the steps of potentising a stereoisomerof said chemical agent, and administering said potentised stereoisomerto the organism.

Another aspect of the invention provides a method of treatment of anorganism suffering from the effects of a chemical agent having one ormore chiral centres, said method comprising the steps of diluting astereoisomer of said chemical agent, and administering said dilutedstereoisomer to the organism.

Still yet another aspect of the invention provides a method of treatmentof an organism suffering from the effects of a chemical agent having oneor more chiral centres, said method comprising the steps of diluting astereoisomer of the chemical agent to an ultra-high dilution of saidstereoisomer, and administering said ultra-high diluted stereoisomer tothe organism.

However, the same effect may be obtained by using dilutions such asthose used in investigations involving hormesis. Such dilutions existbelow the toxic range of a given compound, substance or molecule. Suchdilutions below the toxic range are stimulatory rather than toxic.Usually this phenomenon exists in a narrow range of concentrations justbelow the toxic range.

In the context of the present invention it will be understood by thoseskilled in the art that the term “chemical agent” refers to one or morestereoisomers which induce the effect to be treated in the organism. Thestereoisomer of the chemical agent which may be used to treat theeffects of the chemical agent may be a stereoisomer which induces littleor no effect in the organism of the type to be treated. For instance,where a compound has more than one stereoisomer which induces anundesirable effect and others are less active or inactive, then thestereoisomer which is administered to treat the undesirable effect isselected from the less active or inactive stereoisomers, preferably theenantiomer of the most active stereoisomer of the chemical agent.Alternatively, where all stereoisomers induce the undesirable effect tosome extent it is preferred that the least active stereoisomer isselected although the other less active stereoisomers may also beeffective in treating the undesirable effects of the chemical agent.

The method of the present invention may be used for the treatment of anyorganism, including for example, animals, plants and in particular,humans.

It is well known that chemical compounds which have one or more chiralcentres form stereoisomers. Stereoisomers fall into two broad classes:optical isomers; and geometric (cis-trans) isomers. Optical isomers alsofall into two groups, enantiomers and diastereoisomers. Accordingly,another aspect of the invention is a method of treating an effect of achemical agent, which agent is characterized by one or more chiralcentres, by administering a dilution or an ultra-high dilution orpotentised preparation of a stereoisomer of said chemical agent whereinsaid stereoisomer is selected from the group consisting of enantiomers.While the selection of a less active stereoisomer for use in the methodof the present invention may provide acceptable levels of treatment itis preferred that the enantiomer of the active stereoisomer be selectedwhere possible. It has also been found where an optically activechemical agent is ionic or a salt, such as morphine sulfate, thestereoisomer, enantiomer etc. of the optically active ion or radical maybe derived from other salts of the chemical agent or radical. Forexample, morphine hydrochloride may provide a suitable stereoisomer ofmorphine for the treatment of the effects of morphine sulfate. Thestereoisomer selected to treat the effects of the chemical agent ispotentised.

Where an optically active chemical agent is ionic or a salt, such asmorphine sulfate or nicotine-di-p-toluoyltartrate salt, thestereoisomer, enantiomer etc. of the optically active non-ionic form ornon-salt form of the chemical agent such as morphine or nicotine innon-ionic or non-salt form may be administered in suitably attenuated ordiluted or potentised form. Further, where an optically active chemicalagent is non-ionic or in a non-salt form, such as morphine or nicotinein the non-ionic or non-salt form, the stereoisomer, enantiomer etc. ofthe optically active ionic or salt form such as morphine sulfate ornicotine-di-p-toluoyltartrate salt may be administered in a suitablyattenuated or diluted or potentised form.

Potentization of the stereoisomer may be according to the practices usedin homoeopathy. Preferably the stereoisomer is potentised by succussionor trituration. Attenuation or dilution of the medicinal substance is,in homoeopathy, usually performed in the decimal, centesimal and fiftymillesimal (LM) systems as the standard scales of attenuation, underwhich each successive attenuation contains just {fraction (1/10)},{fraction (1/100)} or {fraction (1/50,000)} as much of the medicinalsubstance as the preceding attenuation. It is preferred that after eachattenuation, the attenuated medicinal substance is succussed (typicallybetween 10 to 100 times at each stage of attenuation) or triturated.Generally, soluble substances may be subjected to succussion andinsoluble or solid substances may be subjected to trituration.

In order to prepare the dilutions or potencies A ml of tincture or Bgrams of medicinal substance are added to C ml or D grams or E parts ofvehicle. Subsequent liquid or solid attenuations are made by serialprogression, succussing or triturating one part of the precedingattenuation to C ml, D grams or E parts of the vehicle respectively. A,B, C, D, and E, are any numbers greater than zero. When preparingconsecutive attenuations, it is not necessary for A, B, C, D or E, to bekept constant. For example for the first, second, third, fourth, fifth,etc. attenuation, the values of A could be A₁, A₂, A₃, A₄, A₅ . . .etc., where A₁, A₂, A₃, A₄, A₅ . . . etc. represent any numbers greaterthan zero. The same principle applies for values of B, C, D, and E.

In the decimal scale of attenuation is generally practised onemillilitre (1.0 ml) of tincture, one millilitre of 1× aqueous solution,or one gram (1.0 g) of 1× trituration represents 0.10 gram of dry crudemedicinal substance. One millilitre of 2× attenuation, or one-gram (1.0g) of 2nd trituration contains 0.01 gram of the dry crude medicinalsubstance. Subsequent liquid or solid attenuations are made by serialprogression, succussing or triturating one (1) part of the precedingattenuation to nine (9) parts of the vehicle, and represent thefollowing proportions of active principle (i.e. dried medicinalsubstance): 2X = 10⁻² 3X = 10⁻³ 4X = 10⁻⁴ 5X = 10⁻⁵ 6X = 10⁻⁶ 7X = 10⁻⁷8X = 10⁻⁸ (and so on . . . )

In decimal attenuations n×=10^(−n) where n is an integer greater than 0.In the case of centesimal attenuations, each attenuation contains justone hundredth of the medicinal substance of the one before, nC=10^(−2n).In the case of fifty millesimal attenuations one millilitre (1 ml) ofthe first fifty millesimal attenuation (1 LM) represents 4.0×10⁻⁹ gramof dry crude medicinal substance. One millilitre (1 ml) of the secondfifty millesimal attenuation (2 LM) represents 8.0×10⁻¹⁴ gram of drycrude medicinal substance. Each subsequent attenuation represents afurther decrease in concentration of dry crude medicinal substance by afactor of 2×10⁻⁵. Each attenuation such as 2×, 3× or n× (2 C, 3 C or nC)(2 LM, 3 LM etc) is generally referred to as a potency.

In order to prepare the solid or liquid stereoisomers of chemicalagents, it is effective to add 2 or more different potencies orattenuations together. A potency refers to a solution, which hasundergone serial dilution and succussion and/or agitation whereasattenuation refers to a process of dilution, which may or may notinvolve succussion or agitation. The term potency also refers to solidattenuations as described herein. The term “different” potencies orattenuations encompasses 2 potencies or attenuations of differentdilutions as well as solutions which have undergone a different numberof steps of serial dilution or attenuation with succussion, or, in thecase of solid attenuations, a different number of steps of serialtrituration as described herein. For example, a person skilled in theart could add the fourth and twelfth potencies or attenuations togetherin equal or unequal quantities. The solution may then be succussed(shaken) N times, where N is any integer greater than zero.Alternatively, the solution is not succussed. Similarly, one could addthe fourth, twelfth, or thirtieth potencies or attenuations together, orany number of combinations of potencies or attenuations.

Also, there is the situation where one could treat a racemic mixturewith both the (+)- and (−)-enantiomers contemporaneously, eithersimultaneously or within the same course of treatment. This could bewith a mixture of equal or unequal volumes of potencies or attenuationsor dilutions or a combination of one or more of same or differentpotencies or attenuations of each enantiomer. This is illustrated in thediscussion and non-limiting examples which follow:

For instance, one could mix one, two, three, four, five or morepotencies, attenuations or dilutions of the (+)-enantiomer to one, two,three, four, five or more potencies of the (−)-enantiomer or vice versa.One or more potencies, attenuations or dilutions of an enantiomer may beprepared in one mixture, and added to an equal or unequal number of thesame or different or any combination of potencies or attenuations of theother enantiomer prepared in a separate mixture. Alternatively, allpotencies, attenuations or dilutions could be added to the same mixture.

(+)- and (−)-enantiomers may be mixed 50:50 or 25:75 or in anyproportion. Thus, a mixture could be prepared by adding 2 ml of 4th,12th and 30th potencies of (+)-enantiomer to 2 ml of 4th, 12th and 30thpotencies of (−)-enantiomer, or vice versa. Alternatively 0.5 ml of 4th,2 ml of 12th and 3.4 ml of 30th potencies could be used, and in fact thenumbers 0.5, 2 and 3.4 could be replaced by any numbers greater thanzero or equal to zero. Also, the 4th, 12th and 30th potencies could bereplaced by any potencies represented by integers greater than or equalto 1. The mixtures of (+)- and (−)-enantiomers prepared separately,could then be administered separately or mixed together. If mixedtogether, the resulting solutions could be succussed or not succussed,and subsequently serially diluted or attenuated or potentised or not.Alternatively, the (+)- and (−)-enantiomers may not be preparedseparately, and mixing could occur in the one container.

Also, in the spirit of the above description, 0.4 ml of 3^(rd) potency,attenuation, or dilution of the (−)-enantiomer, may be added to 1.2 mlof the 13^(th), 5.2 ml of the 41^(st), 4.5 ml of the 200^(th) and 3.7 mlof the 1000th potency, attenuation or dilution. This may then besuccussed or not. In turn, this may then be added to a mixture of 5.1 mlof the 5^(th) potency, attenuation or dilution, 0.3 ml of the 37^(th),and 6.3 ml of the 105^(th) potency, attenuation or dilution of the(+)-enantiomer. This latter mixture may have been succussed or not. Theresulting combination of mixtures may then be serially diluted,attenuated or potentised or not. In this paragraph the number denotingpotencies, attenuations or dilutions can be replaced by any integersgreater than zero. Numbers representing millilitres of potency,attenuation or dilution, can be replaced by any numbers greater than orequal to zero.

(+)- and (−)-enantiomers may be mixed 50:50 or 25:75 or in anyproportion. Thus, a mixture could be prepared by adding 2 g of 4th, 12thand 30th potencies of (+)-enantiomer to 2 g of 4th, 12th and 30thpotencies of (−)-enantiomer, or vice versa. Alternatively 0.5 g of 4th,2 g of 12th and 3.4 g of 30th potencies could be used, and in fact thenumbers 0.5, 2 and 3.4 could be replaced by any numbers greater thanzero or equal to zero. Also, the 4th, 12th and 30th potencies could bereplaced by any potencies represented by integers greater than or equalto 1. The mixtures of (+)- and (−)-enantiomers prepared separately,could then be administered separately or mixed together. If-mixedtogether, the resulting solutions could be succussed or not succussed,and subsequently serially diluted or attenuated or potentised or not.Alternatively, the (+)- and (−)-enantiomers may not be preparedseparately, and mixing could occur in the one container.

Also, in the spirit of the above description, 0.4 ml of 3^(rd) potency,attenuation, or dilution of the (−)-enantiomer, may be added to 1.2 g ofthe 13^(th), 5.2 g of the 41^(st), 4.5 g of the 200^(th) and 3.7 g ofthe 1000th potency, attenuation or dilution. This may then be succussedor not. In turn, this may then be added to a mixture of 5.1 g of the5^(th) potency, attenuation or dilution, 0.3 g of the 37^(th), and 6.3 gof the 105^(th) potency, attenuation or dilution of the (+)-enantiomer.This latter mixture may have been succussed or not. The resultingcombination of mixtures may then be serially diluted, attenuated orpotentised or not. In this paragraph the number denoting potencies,attenuations or dilutions can be replaced by any integers greater thanzero. Numbers representing grams of potency, attenuation or dilution,can be replaced by any numbers greater than or equal to zero.

The same procedures as described above could be used for mixtures ofdiastereoisomers.

The administration of the potentised stereoisomer is typically by anoral route but may be administered intravenously, intramuscularly,transdermally, subcutaneously, intraperitoneally or via any mucousmembrane (typically sublingually). It is particularly preferable toadminister the potentised stereoisomer orally or sublingually. Specificexamples of administration of the potentised stereoisomer includetablets, globuli, liquid dilutions for injection and liquid externalpreparations.

Another method of administering the potentised or attenuatedstereoisomer is to use devices such as the MORA machine, Lisson Machineor Vega Select Machine or other bioresonance or electrodermal testingdevices to detect an electromagnetic or bioresonance signal from thepotency or attenuation and then administer the signal in an unchanged,modified or inverted form to the organism to be treated. These deviceswhich are commercially available, claim to be able to copy the effectsof medicines, dilution or potency and pass the attributes of a medicine,dilution or potency onto a heretofore placebo or medicinally inactivevehicle. The terms “modification” or “inversion” of the signal includeschanging the polarity of the signal.

In order to determine the appropriate potency of the stereoisomer inorder to achieve the most effective treatment of the undesirable effectof the chemical agent it is usually preferred to commence byadministering a low potency of the stereoisomer, say between 1 C-10 Cinclusive (or 1×-10×) and gradually incrementally increasing the potencyuntil the treatment is optimised. Experience may show that 6 C, 15 C, 30C, 200 C are appropriate attenuations in most cases. Attenuation of 1000C, 10,000 C or higher may also produce desirable results. This alsoapplies for non-decimal and non-centesimal potencies.

The vehicles used to attenuate the stereoisomer may be selected from thegroup consisting of water, such as water for injection B.P. or U.S.P.,lactose B.P. or U.S.P., sucrose B.P. or U.S.P. ethanol typically insuitable concentrations (e.g. 15-95%). Also absolute ethanol, purifiedwater, glycerol 85% or other ethanol/water mixture may be used. Othervehicles will also be apparent to those skilled in the art ofhomoeopathy.

The methods of preparation of solid or liquid stereoisomers of chemicalagents into potentised attenuations include where water-soluble oralcohol-soluble isomers are to be prepared into potencies the use ofwater B.P. or purified water alone, or in a mixture of water andethanol, say, 30-45% ethanol. Ethanol-soluble stereoisomers may beprepared using higher concentration ethanol solutions, say 55-95%ethanol or absolute ethanol. It is possible to start using lower andincrementally lower ethanol concentrations as the potency reaches 3 to5× or 3 to 5 C. Final homeopathic liquids often contain 30-40% ethanol.The stereoisomer may be prepared by a process of trituration. Theprocess of trituration is particularly advantageous when thestereoisomer is not readily soluble in water, ethanol or water/ethanolmixes. At potencies beyond 3 C or 6× it is possible to convert fromtrituration to liquid dilutions or vice versa. For example, liquiddilutions may be prepared by first making a trituration and thendiluting it in liquid such as water for injection.

In trituration one part of the medicinal substance, when preparing thefirst potency, or one part of the preceding attenuation when preparingthe second or subsequent potencies, is added to one third of the totalvehicle (e.g. lactose B.P.) used for that potency. The process oftrituration is typically performed with mortar and pestle for 15 to 20minutes. The side of the mortar is then scraped for five minutes todislodge any attenuated substance with the pestle or with a spatula.Then the second third of the vehicle for attenuation is added to themortar and the contents subjected to a further 15 to 20 minutestrituration prior to scraping the sides of the mortar for a further fiveminutes to dislodge attenuated substance. The remaining one third of thevehicle is added to the mortar and the combined mixture is subjected totrituration for a further 15 to 20 minutes to complete the triturationfor that potency. Alternatively, the total vehicle may be added to themedicinal substance or preceding attenuation at each successive stage ofpotency preparation and subjected to 60 minutes of trituration. Eachsuccessive level of attenuation is called a potency.

The process of the present invention may be used to reverse, or inanother embodiment enhance, the effects in vivo, and in vitro, of anyoptically active compounds. Another aspect of the invention provides forthe use of the method of the present invention to enhance the effects invivo and in vitro of any levorotatory compound or to reverse the effectsin vivo and in vitro of any dextrorotatory compound.

Examples of chemical agents which exhibit effects which may be treatedby the present process include a wide variety of physiologically activecompounds such as pharmaceuticals, drugs of addiction, compoundsnaturally occurring in the organism and many others. The process of thepresent invention may be used to alleviate the side effects ofpharmaceuticals, the addictive and undesirable effects of drugs ofaddiction, the toxicity of snake or other animal venoms or the toxiceffects of stereoisomers contained in those venoms; e.g. cobrotoxin,batroxobin, or in general the toxic, physiological or pathologicaleffects of any optically active molecules.

Included in the definition of chemical agents are those stereoisomerswhich undergo or have undergone in vivo transformation into aphysiologically active form. For example, following absorption the drugenalapril is rapidly and extensively hydrolysed to enalaprilat, which isa potent angiotensin converting enzyme inhibitor. Another example is theprodrug atacand, which is rapidly converted to the active drugcandesartan, an angiotensin 2 receptor antagonist by ester hydrolysisduring absorption from the gastrointestinal tract. Dipivefrinehydrochloride is a prodrug which is transformed to liberate adrenaline,an adrenergic agonist into the anterior chamber of the eye.

Pharmaceuticals suitable for use in the present invention includepropranolol. The most common form of propranolol used in medicine is(±)-propranolol hydrochloride (Inderal). This is a β-blocker mainly usedfor the treatment of hypertension or some cardiac arrhythmias. It isavailable in “dextro” (herein referred to as (+)) and “levo” (hereinreferred to as (−)) forms. (+)-propranolol is the inactive form, and hasprobably less than a hundredth the activity of racemic propranolol inblocking isoprenaline-induced tachycardia. In order to reduce theeffects of the (−)-propranolol a potentised attenuation of(+)-propranolol may be used.

On the other hand (+)-propranolol and (±)-propranolol are equallyeffective in abolishing ouabain-induced arrhythmias, while(−)-propranolol has little effect. In order to reduce the effects of(+)-propranolol a potentised attenuation of (−)-propranolol may be used.

Other pharmaceuticals suitable for use in the present invention includetranylcypromine, deserpidine, trimipramine, mianserin, sertraline,paroxetine (and other selective serotonin re-uptake inhibitors),amoxycillin, cobrotoxin, batroxobin, flecainide, sotalol, simvastatin,pravastatin (and other HMG-CoA reductase inhibitors), prednisolone,prednisone, procyclidine, verapamil, (and other calcium channelblockers), capoten (and other angiotension converting enzymeinhibitors), haloperidol, melleril, digitoxin, digoxin, methotrexate(and other optically active cytotoxic or anti-neoplastic compounds),amantadine, cogentin, colchicine, naproxen (and other optically activenon-steroidal anti-inflammatory compounds), warfarin, heparin, ethinylestradiol, venlafaxine hydrochloride, fluoxetine hydrochloride, andsibutramine hydrochloride monohydrate.

The method of the present invention may also be used to treat theundesirable effects of drugs of addiction and the method of the presentinvention may also find use in the treatment of drug addiction ordependency, in particular for the reduction or alleviation of thephysical and psychological effects of drug substances which are commonlyabused. Examples of such drugs of addiction include amphetamines,opiates, cannabinoids, hallucinogens, cocaine and barbiturates. Specificexamples of drugs of this kind include the following drugs and the saltsthereof: morphine, codeine, heroin, phenazocine, Demerol, methadone,barbital, phenobarbital, amobarbital, pentobarbital, secobarbital,Δ⁹-Tetrahydrocannabinol, 11-Hydroxy-Δ⁹-tetrahydrocannabinol,amphetamine, methamphetamine, Lysergic Acid diethylamide (LSD),Scientifically Treated Petrol (STP), or any optically active componentof modern “designer drugs”. Where drugs exist in racemic mixtures, thephysiological effects are counteracted (or enhanced) by administering apotentised attenuation of the enantiomer or less physiologically activeor nonactive stereoisomer. The effects of nicotine addition may becounteracted by preparing a potentised attenuation of a less active ornonactive enantiomer of the active isomer.

The method of the present invention may also be used to counter theeffects of other physiologically active compounds including for example,optically active food additives such as monosodium glutamate or,environmental pollutants. (−)-adrenaline is the active form occurring inhumans and by administering a potentised attenuation of (+)-adrenalinethe tachycardic effects of (−)-adrenaline may be reversed. Similarly,(−)-amphetamine may be used to inhibit the effects of pharmaceuticaldoses of (+)-amphetamine such as insomnia or vice versa. Also,(+)-ephedrine may be used to inhibit the effects of pharmaceutical dosesof (−)-ephedrine or vice versa.

Another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterised by one or more chiralcentres for the preparation of a medicament for the treatment of acondition characterised by the effects of said chemical agent.

Another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the treatment of thetoxic, physiological and/or pathological effects of said chemical agent.

Yet another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the alleviation of theside effects of pharmaceuticals.

Still yet another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the treatment of theaddictive and other undesirable effects of drugs of addiction.

Still yet another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the alleviation of thephysical and psychological effects of drugs of addiction.

Still yet another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the treatment of thetoxic effects of animal venoms.

Another aspect of the invention is the use of a dilution or anultra-high dilution or potentised preparation of a stereoisomer of achemical agent, which agent is characterized by one or more chiralcentres for the preparation of a medicament for the treatment of thetoxic effects of snake venoms.

The present invention is further described by the following non-limitingexamples.

EXAMPLE 1 Homeopathic Treatment of Mice Administered PropanololHydrochloride

BALB/C mice are injected with the LD₅₀ of propranolol hydrochloride.This drug is a β-blocker which has been used to treat blood pressure andsometimes migraine. Students also use propranolol to decrease sweatingand tremor in examinations where it actually promotes a relaxed feeling.

The LD₅₀ is the dose at which 50% of mice will die. The propranolol hasthe effect of slowing the heart rate with resultant unconsciousness anddeath. The LD₅₀ for mice is 565 mg/kg (orally), 22 mg/kg IV, andapproximately 150 mg/kg intraperitoneally.

Half of the mice receive a homeopathic and the other half receive anindistinguishable placebo. Mice are analysed to determine if those inthe treated group exhibit greater survival than those in the placebogroup.

EXAMPLE 2 Inhibition of (S)-(−)-Propranolol Hydrochloride by itsEnantiomer in White Mice

Methods

This experiment insofar as the procedure of anaesthesia is concerned,was a modification of the procedure used for determining pentobarbitonesleeping time described by Lovell. Seventy-seven placebo ICRconventional mice and seventy-seven treatment group ICR conventionalmice were utilized which gave the experiment an 80% power and test levelof 5%. This assumed 64% survival in the treatment group and 40% survivalin the placebo group. Equal numbers of male and female mice wereallocated alternately to 2 groups called “A” and “B”. A coin was thentossed to determine which of A or B would be the treatment group.

The following is a table of weight summary statistics.

Pilot Analysis with Weight

Summary Statistics of Weight (Grams) ARM Mean Std N 1 24.78 2.70 76 224.51 2.31 72 All 24.65 2.51 148In the above table1 = weight of medicine mouse and2 = weight of placebo mouse.All exclusions from the total sample were in accordance with prospectivecriteria.

To make the homeopathic potency for this experiment one hundred mg of(R)-(+)-propranolol HCl obtained from Fluka Chemie, Switzerland, ≧99.9%pure, was added to 5 ml 90% ethanol B.P. obtained from Sigma ChemicalCompany, Clayton, Victoria, Australia, in a brown tinted 20 ml glassbottle with dropper and rubber bulb, obtained from Plasdene Glaspak,Victoria, Australia. The bottle was closed and was given 20 forcefuldownward succussions by hand at the rate of ½-1 Hz. This was the firstpotency. Three drops from this bottle were added to a second identicalbottle containing 9 ml of 90% ethanol B.P. (40 drops 90% ethanol wasapproximately 1 ml.) The second bottle was given 20 downward succussionsat ½-1 Hz. This was the second potency. Three drops from the secondpotency were placed into a third bottle containing 9 ml ethanol andsuccussed as previously to produce the 3^(rd) potency and so on untilthe 29^(th) potency was produced. This was the potency used in theexperiment. Therefore, the potentised propranolol used in thisexperiment was diluted by a factor of greater than 10⁵². This is well inexcess of Avogadro's number (6.023×10²³).

Indistinguishable placebo was prepared as above, but instead of 100 mgisomer being added to the first bottle, 3 drops of 90% ethanol wereadded to a bottle containing 5 ml 90% ethanol B.P. The 90% ethanol usedfor preparation of placebo came from the same bottle of ethanol, whichwas used for preparation of the homeopathic medicine. Subsequent“potencies” of placebo were succussed as described above for the isomerpotencies until the 29^(th) potency of placebo was prepared. This wasthe indistinguishable placebo, which was used in the experiment.

Mice were bred in a standard non-pathogen free animal house andtransferred to the laboratory animal house (temperature 20-22° C.,photoperiod 7 am to 7 pm, 1 week before the experiment to allowacclimatization. During breeding mice were fed their standard diet. Theinventors preference was to use non-irradiated Mouse Maintenance Diet(RM1 expanded) (Special Diets Services Ltd). During breeding mice werefed non-irradiated Mouse Maintenance Diet (RM3 expanded). However, dueto non-availability of this product a standard mouse diet for miceavailable at the Institute of Zoology was used. The only investigatorblinded during this experiment was the anesthetist. This was consideredto be the most likely step in the experiment at which bias could beintroduced.

At 4.30 pm on the day before the experiment, mice were given treatmentand placebo fluids using disposable non-heparinized hematocrit capillarytubes (75 mm/75 microlitre, Hirschmann Laborgeräte). For this purpose amouse was taken from its cage by an experienced mouse handler and heldin a supine position. Using a hematocrit tube, this person removedapproximately 0.05 ml of the respective medicine or placebo from thestorage test tubes (containing medicine or placebo). The filledhematocrit tube was introduced just behind the incisors of the mousesuch that it was impelled to drink quickly. The tube was removed as soonas possible and the mouse was held for another 20 seconds in the supineposition. Each mouse was then returned to its cage. Mice in fact seemedkeen to drink both treatment and placebo fluids and generally thehematocrit tube just needed to be presented close to the incisors, andthe mice then drank readily from them.

Each cage had a constant amount of unautoclaved softwood (pine) beddingjust covering the cage floor. Cages were made out of plastic with a wiresee-through roof. The drinking water bottle and food was not placed inthe cage until 10 minutes later. This same procedure was repeatedbetween 9.00-9.30 am on the morning of the experiment, with theexception that prior to giving each mouse it's respective medicine orplacebo, the mouse was placed in a clean strong plastic bag andsuspended from a Pesola 30 g or 60 g scale (Pesola, Switzerland,www.pesola.ch), to measure weight in grams. Mice were allowed food andwater ad libitum at all stages of the experiment up until just beforethe injection of anaesthetic, with the exception of the brief period ofabstinence (10 minutes) after giving oral liquids by capillary pipette.

To prepare the homeopathy used each day in the experiment, 5 drops ofthe 29^(th) potency prepared as described above were added to 15 mls ofwater in a 20 ml Hamilton Laboratory Glass test tube with a glassstopper. The test tube was not filled more that two-thirds with liquid.The test tube was given 20 forceful downward succussions or shakes.Black paper was placed around the test tube to protect it from light.The placebo specimen was prepared in the same way. All glassware such astest tubes used recurrently during the experiment was washed with tapwater, copiously rinsed with distilled water, hot-air dried and finallyheated for 2 hours at 200° C. before being considered clean and reusable(Blackie Foundation Trust). The same glassware was used for treatmentand placebo preparation. In addition, (see Kuzeffet al (reference 12)and Kuzeffet al (2) (reference 13), cages and water bottles were washedafter each experiment with “Pur+Aloe Vera” (pH neutral, Henkel, Austria,245604), mixed 50:50 with Belina ACE (Greece). Both these products arecommercially available in Sofia.

Between 12 and 16 mice were selected each afternoon about 4.30 pm. Theywere divided into two groups of 6-8 and each group was placed in aplastic cage approximately 24 cm×14.5 cm with walls 14 cm high, withstainless steel mesh roof, cradle for inserting a water bottle, andgrill onto which could be placed dry food for ad libitum consumption. Atthe start of the experiment only 12 mice were tested each day. As theexperimenters became accustomed to the procedure this number wasincreased to 16. Mice were administered potency or placebo according tothe method described in the next paragraph, which relates the conduct ofthe experiment on the morning of the next day.

The experiment started when a mouse was removed from its cage between10-11 am in the morning, and held gently but firmly in the supineposition. It was injected i.p. (intraperitoneal) with a solution of 2%Xylazine hydrochloride (Rometar, Spofa, Prague) diluted 50:50 in Normalsaline B.P. for injection. The volume of this solution administered tomice intraperitoneally was 0.1 ml/10 gm body weight. Injections weregiven using a 0.5 ml Becton-Dickinson insulin syringe with 27 gaugeneedle, intraperitoneally into the left lower quadrant of the mouse'sabdomen by an experienced anaesthetist. After injection the mice wereheld in the supine position for approximately 2-5 minutes until asleepor heavily sedated. Each mouse was then placed into an individualplastic mouse cage with softwood bedding but no lid. They remained hereuntil the process of administration of oral potency or placebo wasfinished. After this they were returned to their communal group A orgroup B cage which had ad libitum food and water available. The numberof mice alive at 9 am the next morning was the end point of theexperiment.

S-(−)-Propranolol HCl was dissolved in sufficient Normal Saline B.P.such that injecting 0.1 ml/10 gm body weight of the solutionintraperitoneally would give a dose of isomer of 155 mg/kg. TheS-(−)-propranolol diluted in Normal Saline for Injection B.P. in thismanner is not very soluble. Dissolution was assisted by placing the 10ml Hamilton Laboratory Glassware test tube containing the Normal Salineand (S)-(−)-Propranolol HCl into a beaker containing warm tap water atapproximately 40° C.

Treatment and placebo mice were alternately presented in random order tothe anaesthetist who was blinded to the sequencing. This means that onsome of the days of the experiment a placebo mouse was presented firstfor Rometar injection and on other days it was a treatment mouse. Thiswas done for both Rometar and (S)-(−)-propranolol injections.

Five minutes after injection of Rometar the mice were heavily sedated orasleep. It was at this point that treatment and placebo mice wereinjected with (S)-(−)-propranolol HCl in alternate sequence, in the sameorder in which they were injected with Rometar. After all mice hadreceived i.p. (S)-(−)-propranolol HCl, they were administered oraltreatment or placebo in the same sequence in which they had receivedtheir injections. According to the protocol each mouse was then to begiven 1 drop of potentised isomer or placebo, at approximately 5, 10,15, and 20 minutes after (S)-(−)-propranolol HCl injection, and also at30, 40, 50, 80 and 110 minutes.

Results

It should be noted that the dose of (S)-(−)-propranolol HCl injected inthis experiment was started at 107 mg/kg intraperitoneally and by trialand error on successive days it was determined that the LD50 of(S)-(−)-propranolol HCl i.p. was approximately 155 mg/kg. In the courseof the experiment batches of mice were at various times also injectedwith 214 mg/kg i.p. and 180 mg/kg i.p.

The trend for survival was in favour of homeopathy with somewherebetween 9% and 24% more mice surviving than placebo mice. Mouse recoverywas substantially faster in the treatment mice than in the placebo mice.

EXAMPLE 3 Inhibition of (S)-(−)-Propranolol Hydrochloride by itsEnantiomer in White Mice (2)

Methods

ICR conventional mice were used in this experiment. The experiment wasperformed between December 2001 and April 2002 using 3 batches of mice.254 placebo mice and 254 treatment group mice were used in thisexperiment. The following is a table of weight summary statistics.

Double-Blind Analysis with Weight

Summary Statistics of Weight (Grams) ARM Mean Std N m 23.20 3.99 238 p23.48 4.55 246 All 23.34 4.29 484In the above tablem = weight of medicine mouse andp = weight of placebo mouse.All exclusions from the total sample were in accordance with prospectivecriteria.

Placebo mice mean was 23.50 gm SD=4.51, n=254. As discussed in Example2, assuming that the real difference in survival was somewhere between9% and 24%, a sample of approximately 500 mice, 250 treatment and 250placebo, assuming p=0.05 with 17% improved survival in the treatmentgroup, i.e., 50% survival in the treatment group and 33% survival in theplacebo group, gives a power of 0.95 for a two-sided test, and 0.98 fora one-sided test (Hennekens et al (1987)).

Homeopathy was prepared fresh every 2 days as follows. Twenty mg(R)-(+)-propranolol hydrochloride, obtained from Fluka Chemie,Switzerland, ≧99.9% pure, was added to two mls 40% ethanol B.P. in a 10ml Hamilton Laboratory Glass test tube with a glass stopper (HamiltonLaboratory Glass Ltd). This is neutral glass type one in the EuropeanPharmacopoea. Twenty forceful downward succussions were given to thefluid in the test tube, in a vertical line at a rate of between one-twoHertz. A non-heparinised disposable hematocrit capillary tube (75 mm/75microlitre, Hirschmann Laborgeräte) was used to add 3 drops of thissolution to another test tube containing 7 ml 40% ethanol B.P.—ten mlHamilton Laboratory Glass test tubes were again used (40 drops, 40%ethanol, approximately=1 ml).

Note that the test tube should not be more than two-thirds full, i.e.,there must be plenty of room in the test tube for the fluid to collideviolently with the test tube walls when it is succussed. This secondtest tube was succussed 20 times at one-two Hertz. Three drops of thispotency were added to a third 10 ml Hamilton test tube containing onceagain 7 ml 40% ethanol B.P. The same process was repeated until the29^(th) dilution or potency was reached. The 30^(th) potency wasprepared from the 29^(th) in the same manner. The 29^(th) potency wasstored in a dark place and wrapped in paper for use the next day. Afresh 29^(th) potency was prepared every 2 days.

Indistinguishable placebo was prepared by adding 3 drops from ahematocrit capillary tube to a 10 ml test tube containing 7 ml 40%ethanol B.P. and giving 20 succussions as above described for themedicine preparation. All glassware such as test tubes used recurrentlyduring the experiment was washed with tap water, copiously rinsed withdistilled water, hot air dried and finally heated for 2 hours at 200° C.before being considered clean and reusable (Blackie Foundation Trust).The same glassware was used for treatment and placebo preparation. Inaddition, (see Kuzeffet al (reference 12) and Kuzeffet al (2) (reference131 cages and water bottles were washed after each experiment with“Pur+Aloe Vera” (pH neutral, Henkel, Austria, 245604), mixed 50:50 withBelina ACE (Greece). Both these products are commercially available inSofia.

It should be noted that 40% ethanol produces a type of froth whensuccussed in this way. The rate of succussion was performed to besufficiently slow to enable this froth to settle down substantiallybetween each individual succussion. The indistinguishable placeboconsisted of 7 ml 40% ethanol B.P. added to an indistinguishableHamilton Laboratory Glass test tube. This was given 20 succussions asabove described for the medicine preparation.

Prior to the commencement of the experiment at 9.30 am each day, anindividual who did not participate in the actual conduct of theexperimental protocol, other than performing randomisation, took theindistinguishable test tubes containing potency and placebo into aclosed room. One sticky label was marked with the letter “A” and anothersticky label was marked with the letter “B”. A coin was tossed todetermine if A or B would correspond to medicine or placebo. The testtube containing potency was wrapped with paper and labeled with thesticker, “A” or “B”, indicated by randomization. The placebo-containingtest tube was wrapped with indistinguishable paper in the same way asthe potency-containing test tube, and again the appropriate label wasaffixed according to randomisation. The blind randomization code waskept in the personal possession of the randomiser until after theexperiment and not revealed to any other person.

Mice were bred in a standard animal house and transferred to thelaboratory animal house (temperature 20-22° C., photoperiod 7 am to 7pm, 1 week before the experiment to allow acclimatization. Duringbreeding mice were fed their standard diet. In the past this had beennon-irradiated Mouse Maintenance Diet (RM1 expanded) (Special DietsServices Ltd). During breeding mice were fed non-irradiated MouseMaintenance Diet (RM3 expanded) (Walker et al, 1993). However, due tonon-availability of this product, a standard mouse diet available at theInstitute of Zoology was used.

Treatment and placebo fluids were given to mice using disposablehematocrit capillary tubes referred to (75 mm/75 microlitre, HirschmannLaborgeräte). For this purpose a mouse was taken from its cage by anexperienced mouse handler and held in a supine position. This personremoved approximately 0.05 ml of the respective blinded and randomisedmedicine or placebo from the storage test tubes (containing medicine orplacebo). The filled hematocrit tube was introduced just behind theincisors of the mouse such that it was impelled to drink quickly. Thepipette was removed as soon as possible and the mouse was held foranother 20 seconds in the supine position. Each mouse was then returnedto its cage.

Each cage had a constant amount of unautoclaved softwood (pine) beddingjust covering the cage floor. Cages were made out of plastic with a wiresee-through roof. The drinking water bottle and food were not placed inthe cage until 10 minutes later. This same procedure was repeatedbetween 9.00-9.30 am on the morning of the experiment, with theexception that prior to giving each mouse it's respective medicine orplacebo, the mouse was placed in a clean strong plastic bag andsuspended from a Pesola 30 g or 60 g scale (Pesola, Switzerland,www.pesola.ch), to measure weight in grams. Mice were allowed food andwater ad libitum at all stages of the experiment up until just beforethe injection of anaesthetic, with the exception of the brief period ofabstinence (10 minutes) after giving oral liquids by capillary tube.

Sixteen mice were selected each afternoon about 4.30 pm. They weredivided into two groups of 8 and each group was placed in a plastic cageapproximately 20 cm×30 cm with walls 20 cm high, with wire meshsee-through roof, cradle for inserting a water bottle, and grill ontowhich could be placed dry food. A coin was tossed to determine whichgroup would be called “group A”, and the other group was called “groupB”. A coin was tossed again (unnecessarily since test tubes A and B werealready randomised), to determine if group A mice would be allocated toreceive test tube A or test tube B in the experiment. Group B mice thenreceived the opposite. Mice were then given randomised and blindedpotency or placebo as described in the paragraph below which relates theconduct of the experiment on the morning of the following day.

Two investigators and two laboratory assistants were involved in theperformance of the experiment and a third investigator performedrandomisation and blinding only. The four mouse handlers were dividedinto two groups of two handlers for the experiment on each day. This wasdone by placing the numbers 1-4 on each of 4 identical cards which wereplace in a box which was shaken. The cards were then withdrawn blind byeach investigator. Investigators who in this way selected the numbersone or two were paired for the experiment and handled the “group A” ofmice. Those who extracted numbers 3 and 4 handled the “group B” of miceon a given day of the experiment. This was an extra precaution todecrease the risk of systematic biases being introduced in the handlingof the animals, although strictly speaking it was not necessary in arandomised and blinded experiment. The experiment started when a mousewas removed from its cage between 10-11 am in the morning, and heldgently but firmly in the supine position. It was injected i.p. with asolution of 2% xylazine hydrochloride (Rometar, Spofa, Prague) diluted50:50 in physiological saline for injection B.P. The volume of thissolution administered to mice intraperitoneally was 0.1 ml/10 gm bodyweight. Injections were given using a 0.5 ml insulin syringe with 27gauge needle, intraperitoneally into the left lower quadrant of themouse's abdomen by an experienced anaesthetist. After injection the micewere held in the supine position for approximately 2-5 minutes untilasleep or heavily sedated. Each mouse was then placed into an individualplastic mouse cage with softwood bedding but no lid. They remained hereuntil the process of administration of oral potency or placebo wasfinished. After this they were returned to their communal group A orgroup B cage which had ad libitum food and water available. The numberof mice alive at 9 am the next morning was the end point of theexperiment. It was estimated that under the conditions of thisexperiment the LD50 of (S)-(−)-propranolol hydrochloride injectedintraperitoneally into white ICR mice was approximately 155 mg/kg. Thiswas determined over a few days by trial and error starting at a dose of107 mg/kg i.p. as suggested elsewhere.

(S)-(−)-Propranolol HCl was dissolved in sufficient Normal Saline B.P.such that injecting 0.1 ml/10 gm body weight of the solution would givea dose of isomer of 155 mg/kg. Treatment and placebo mice werealternately presented to the anaesthetist. The (S)-(−)-Propanololdiluted in Normal Saline for Injection B.P. in this manner is not verysoluble. Dissolution was assisted by placing the 10 ml HamiltonLaboratory Glassware test tube containing the Normal Saline and(S)-(−)-Propranolol HC into a beaker containing warm tap water atapproximately 40° C.

Five minutes after injection of Rometar the mice were heavily sedated orasleep. It was at this point that the mice were injected with(S)-(−)-propranolol HCl in alternate sequence, in the same order inwhich they were injected with Rometar. Mice which died prior toinjection of (S)-(−)-propranolol were excluded according to theprospective design. After all mice had received i.p. (S)-(−)-propranololHCl, they were administered oral treatment or placebo in the samesequence in which they had received their injections. Each mouse wasgiven 1 drop of potentised isomer or placebo, according to randomisationand blinding, at approximately 5, 10, 15, and 20 minutes after(S)-(−)-propranolol HCl injection, and also at 30, 40, 50, 80 and 110minutes.

Results

It should be noted that the dose of (S)-(−)-propranolol HCl injected inthis experiment was started at 130 mg/kg intraperitoneally. Over theensuing 6 months we attempted to maintain with varying successapproximately 50% mortality in the treatment mice. Doses of 130 140 and155 microgram/kg were injected intraperitoneally. The LD50 for the miceseemed to vary somewhat perhaps depending on season and the weight andage of the batches of mice which were being tested. As already mentionedthis experiment was performed in 3 batches between 86 to 240 mice. Notethat in the analysis “medicine” refers to dose of intraperitoneal(S)-(−)-propranolol hydrochloride which was injected and not to thehomeopathic medicine which was potenitized (R)-(+)-propranololhydrochloride. The homeopathic medicine is referred to as “treatment”.

The end-point for statistical analysis was the difference in survivalbetween the placebo and treatment mice at 9 am on the morning after theywere injected. 254 treatment and 254 placebo mice were admitted to theexperiment. Twenty-four mice in total died after injection ofintraperitoneal rometar and before injection of intraperitonealisomer—sixteen treatment mice and eight placebo mice. The exclusion ofthese mice was in accordance with prospective criteria. Prospectively itwas anticipated to perform a Chi-square test on the entire body ofrandomised data, but a logistic regression analysis was also done, totake into account weight of mice and dosage of intraperitoneal(S)-(−)-propranolol hydrochloride which was injected. Statisticalanalysis was performed by Statistical Investigations Pty Ltd., and asummary of the analysis follows as it was presented by the statisticalconsultant:

Chi-Square Tests

Cross-Tabulation of ‘Arm’ vs ‘a-d’: TABLE 1 Alive Dead Odds OR Medicine117 121 0.97 1.52 Placebo 96 150 0.64 1 Total 213 271Under the null hypothesis that:

-   -   the proportions of alive and dead are the same for medicine and        placebo        the Chi-Square=5.0429 with 1 degree of freedom, which has a        probability of 0.0247. So we reject the null hypothesis at the        0.05 significance level.        Logistic Models

Logistic regression models were used to express the relationship betweena dichotomous outcome (alive/dead) and the experimental factors: ‘arm’(medicine/placebo), ‘(−)-prop’ (dose of (−)-propranolol hydrochlorideinjected i.p. in mg/kg), and weight category (Low, Medium, or High). Theweight categories are defined by the 33.3 percentile (21.5 g) and 66.7percentile (24.0), i.e. Low: ≦21.5 g, Medium: 21.6 g-24.0 g and High:≧24.1 g.

A series of models were fitted starting with a saturated model.Subsequent steps in the search for the best model involved fittingadditional models that represented the successive removal of terms fromthe saturated model. At each step a statistical test (‘likelihood ratiotest’) was carried out to see if there was a statistically significantdifference between the models. The process was stopped when there was asignificant difference between models, because this indicated that thelast term that was removed significantly reduced the fit of the model.

The result of this model fitting process is a model that can besummarised as follows (degrees of freedom in brackets):

-   -   Log of the odds of        survival=intercept(1)+arm(1)+treatment(2)+weight(2)+weight*treatment(4)

Parameter estimates for this model are: TABLE 5 Standard Parameter DFEstimate Error Chi-Square Pr > ChiSq Intercept 1 −0.0240 0.1127 0.04540.8313 ARM (medicine) 1 0.2072 0.0945 4.8112 0.0283 (-)-prop (130 mg/kg)1 0.1438 0.1490 0.9313 0.3345 (-)-prop (140 mg/kg) 1 0.0654 0.17920.1333 0.7150 Weight (Low) 1 −0.0515 0.1486 0.1200 0.7290 Weight(Medium) 1 −0.2445 0.1485 2.7096 0.0997 (-)-prop (130 mg/kg) by Weight(Low) 1 −0.0833 0.1961 0.1806 0.6709 (-)-prop (130 mg/kg) by Weight(Medium) 1 −0.1760 0.1969 0.7985 0.3715 (-)-prop (140 mg/kg) by Weight(Low) 1 −0.5236 0.2187 5.7315 0.0167 (-)-prop (140 mg/kg) by Weight(Medium) 1 −0.0340 0.2325 0.0214 0.8837(-)-prop = dose of (-)-propranolol HCL injected intraperitoneally inmg/kg

Under the null hypothesis (parameter=0) the square of ratio of theparameter estimate to its standard error has a Chi-Square distribution(I degree of freedom). In the above table the ‘ARM (medicine)’ factorand ‘(−)-prop (140 mg/kg) by Weight (Low)’ interaction have P-valuesless than 0.05, thus in both cases we reject the null hypothesis of zeroparameter value.

As there is a weight by i.p. (−)-propranolol interaction in this model,an odds ratio can only be calculated for the arm factor. It is 1.51 witha 95% CI of for 1.045 to 2.19. This odds ratio estimate shows that thetreatment mice were 1.5 times more likely to survive than placebo mice.

The end-point for statistical analysis was the difference in survivalbetween the placebo and treatment mice. The odds ratio estimates showthat the treatment mice were 1.5 times more likely to survive thanplacebo mice. This was statistically significant with p=0.025 with ChiSquare test and 0.028 using the logistic regression model. Elevenpercent more treatment mice survived than placebo mice.

EXAMPLE 4 Inhibition of (−)-Trans-(1S, 2S)-U-50488 by its Enantiomer inWhite Mice—A Blind Randomised Placebo-Controlled Study

Homeopathy was prepared as follows. Ten mg (+)-trans-(1R,2R)-U-50488 wasadded to one ml distilled water in a 10 ml Hamilton Laboratory Glasstest tube with a glass stopper (Hamilton Laboratory Glass Ltd). This isneutral glass type one in the European Pharmacopoeia. Twenty forcefuldownward succussions were given to the fluid in the test tube, in avertical line at a rate of between one-two Hertz. A non-hepariniseddisposable 40 drops in 1 ml hematocrit capillary tube (75 mm/75microlitre, Hirschmann Laborgeräte) was used to add 3 drops of thissolution to another test tube containing 7 ml 40% ethanol B.P. Ten mlHamilton Laboratory Glass test tubes were used again (40 drops 40%ethanol approximately =1 ml).

The test tube was not more than two-thirds full, i.e., there must beplenty of room in the test tube for the fluid to collide violently withthe test tube walls when it is succussed. This second test tube wassuccussed 20 times at one-two Hertz. Three drops of this potency wasadded to a third 10 ml Hamilton test tube containing once again 7 ml 40%ethanol B.P. The same process was repeated until the 29^(th) dilution orpotency was reached. The 30^(th) potency was prepared from the 29^(th)in the same manner. Potencies were stored in a dark place and wrapped inpaper for use on later days. They were stored in the dark away fromdirect sun and sources of electromagnetic energy such as power cables,computers, television, metal surfaces, refrigerators and otherelectrical equipment. The potencies were not stored near substances withstrong smells. Indistinguishable placebo was prepared by adding 3 dropsof 40% ethanol from a hematocrit capillary tube to a 10 ml test tubecontaining 7 ml 40% ethanol BP and giving 20 succussions as per thetreatment preparation. All glassware such as test tubes used recurrentlyduring the experiment was washed with tap water, copiously rinsed withdistilled water, hot air dried and finally heated for 2 hours at 200° C.before being considered clean and reusable (Blackie Foundation Trust).The same glassware was used for treatment and placebo preparation. Inaddition, (see Kuzeff et al and Kuzeff et al (2), cages and waterbottles were washed after each experiment with “Pur+Aloe Vera” (pHneutral, Henkel, Austria, 245604), mixed 50:50 with Belina ACE (Greece).Both these products are commercially available in Sofia.

Homeopathy for use in this experiment was prepared as follows: 1.5 ml ofliquid was removed from the tube with the 4^(th) potency and added to a10 ml Hamilton Laboratory test tube. Likewise 1.5 ml was removed fromthe 12^(th) potency and added to the same tube. Then 1.5 ml was removedfrom the 30^(th) potency and added to the tube. The contents were given20 forceful downward succussions at 1-2 Hertz to prepare the potency H1.This is the mixture, which was used in the experiments. It is importantthat the mixture of the 4^(th), 12^(th) and 30^(th) potencies occursquickly, eg. within 30 seconds, so that the tube can be givensuccussions quickly.

To make the potency for use in the experiment 3 drops of H1 is added toa test tube containing 7 ml 40% ethanol. This is immediately given 20succussions at 1-2 Hz to produce H2. The test tube was then wrapped withwhite paper. This is the final preparation which was administered to themice.

Placebo Preparation

The placebo was made immediately before the medicine every morning. Thiswas done to try to avoid contamination of the placebo with the potency.The indistinguishable placebo was made by adding 3 drops of 40% ethanolfrom a capillary hematocrit tube to a test tube containing 7 ml 40%ethanol B.P. added to an indistinguishable Hamilton Laboratory Glasstest tube. This was given 20 succussions as above described for themedicine preparation. This was made fresh daily. The test tube was thenwrapped with white paper.

It should be noted that 40% ethanol produces froth when succussed inthis way. The rate of succussion was performed to be sufficiently slowto enable this froth to settle down substantially between eachindividual succussion.

Before the start of the experiment at 9:30 am each day, an individualwho did not participate in the conduct of the experimental protocol,other than performing randomisation, took the indistinguishable testtubes into a closed room. The placebo was made first every morning andafter that the potency. This was to avoid contamination of the placebo.Stickers were available named “A” and “B”. After the placebo was made acoin was tossed to decide if the medicine or placebo was going to becalled “A” or “B”. The placebo was then wrapped with paper with thesticker “A” or “B” stuck onto it according to the randomisation. Thepotency or medicine-containing test tube was wrapped withindistinguishable paper in the same way as the placebo-containing testtube and again the appropriate label was affixed according torandomisation. The blind randomisation code was kept in the personalpossession of the randomiser until after the experiment and not revealedto any other person.

Mice were bred in a standard animal house and transferred to thelaboratory animal house (temperature 20-22° C., photoperiod 7 am to 7pm, 1 week before the experiment to allow acclimatisation. Duringbreeding mice were fed their standard diet. This consisted of a standardmouse diet available at the Institute of Zoology.

Treatment and placebo fluids were given to mice using the disposablehematocrit capillary tubes. (This was done after selecting the newgroups of mice at 4.30 pm each day and also at about 9.30 am the nextmorning. To give oral fluids a mouse was taken from its cage by anexperienced mouse handler and held in a supine position. This personremoved approximately 0.05 ml of the respective blinded and randomisedmedicine or placebo from the storage test tubes (containing medicine orplacebo). The filled hematocrit tube was introduced just behind theincisors of the mouse such that it was impelled to drink quickly. Thepipette was removed as soon as possible and the mouse was held foranother 20 seconds in the supine position. Each mouse was then returnedto its cage. The drinking water bottle and food were not placed in thecage until 10 minutes later. This same procedure was repeated between9.00-9.30 am on the morning of the experiment, with the exception thatprior to giving each mouse its respective medicine or placebo, the mousewas placed in a clean strong plastic bag and suspended from a Pesola 30g or 60 g scale (Pesola, Switzerland, www.pesola.ch), to measure weightin grams. Mice were allowed food and water ad libitum at all stages ofthe experiment up until just before the injection of anaesthetic, withthe exception of the brief period of abstinence (10 minutes) aftergiving oral liquids by capillary tube.

Each cage had a constant amount of unautoclaved softwood (pine) beddingjust covering the cage floor.

Mouse Selection

Eighteen mice were selected each afternoon about 4.30 pm except day onewhen 16 were selected. All mice were placed in one cage. A mouse wasremoved from this cage and a coin was tossed to decide randomisation togroup “A” or group “B”. As soon as 9 mice were in either group “A” orgroup “B”, then the remaining mice of the eighteen selected wereallocated to the group which did not contain 9 mice yet. The group of 16on day one was treated analogously. Group “A” and group “B” mice werehoused in a plastic cage approximately 24 cm×14.5 cm with walls 14 cmhigh, with stainless steel mesh roof, cradle for inserting a waterbottle, and grill onto which could be placed dry food.

A coin was tossed again to determine if group A mice would be allocatedto receive test tube A or test tube B in the study. Group B mice thenreceived the opposite.

Two investigators and two laboratory assistants were involved in theperformance of the study and a third investigator performedrandomisation and blinding only. The four mouse handlers were dividedinto two groups of two handlers for the experiment on each day. This wasdone by placing the numbers 1-4 on each of 4 identical cards, which wereplaced in a box, which was shaken. Each investigator then withdrew thecards blind. Investigators who in this way select the numbers one or twowere paired for the experiment and handled “group A” mice. Those whoextracted numbers 3 and 4 handled the “group B” mice on a given day ofthe experiment.

The next part of the experiment started when a mouse was removed fromits cage between 10-11 am in the morning, and held gently but firmly inthe supine position. It was injected i.p. with a solution (−)-U50488diluted in normal saline for injection B.P. During the first 3 days 52mice of the total 210 were used to estimate the LD50 of (−)-U50488 whichis approximately 25 mg/kg i.p. This left 158 mice available foranalysis. The volume of this solution administered to miceintraperitoneally was 0.1 ml/10 gm body weight. Injections were givenusing a 0.5 ml insulin syringe with 27-gauge needle, intraperitoneallyinto the left lower quadrant of the abdomen by an experiencedanaesthetist. After injection the mice were held in the supine positionfor approximately 2-5 minutes until asleep or heavily sedated. Eachmouse was then placed into an individual plastic mouse cage withsoftwood bedding but no lid. They remained there until the process ofadministration of oral potency or placebo was finished. After this theywere returned to their communal group A or group B cage, which had adlibitum food and water available. The number of mice alive at 9 am thenext morning was the end point of the experiment.

After all mice had received i.p. (−)-trans-(1S, 2S)-U-50488, they weregiven oral treatment or placebo in the same sequence in which they hadreceived their injections. Each mouse was given 1 drop of potentisedisomer or placebo orally by capilliary hematocrit tube, according torandomisation and blinding, at approximately 5, 10, 15, and 20 minutesafter (−)-propranolol HCl injection, and also at 30, 40, 50, 80 and 110minutes.

Results

Approximately 19.5% more treatment mice survived than placebo mice afteradministration of the LD50.

Cross-Tabulations

The body weight categories are defined by the 33.3 percentile (22.5 g)and 66.7 percentile (25.0), i.e. Low: ≦21.5 g, Medium: 21.6 g-24.0 g andHigh: ≧24.1 g.

It can be seen in the following cross-tabulations that some of the cellshave low or zero frequencies. TABLE 1 Table 1 of i.p.U50488 byBodyWeight Controlling for ARM = homeop BodyWeight IPU50488 LO MED HITotal 10 2 2 4 8 20 1 3 14 18 25 36 22 21 79 Total 39 27 39 105

TABLE 2 Table 2 of IPU50488 by BodyWeight Controlling for ARM = placeboBodyWeight IPU50488 LO MED HI Total 10 0 4 4 8 20 3 8 7 18 25 31 30 1778 Total 34 42 28 104

These low frequency cells stop the estimation method used in logisticregression (method of maximum likelihood) from converging. This in turnleads to highly imprecise and biased estimates. In order to getconvergent parameter estimates the following logistic model is fittedonly to i.p U50488=25 mg/kg, only. This was the estimated LD50 and is inaccordance with the prospective study design.

Logistic Models

Logistic regression models were used to express the relationship betweena dichotomous outcome (alive/dead) and the experimental factors: ‘arm’(medicine/placebo), and body weight category (Low, Medium, or High).

Two models were fitted. The first contained an interaction between bodyweight and arm and represents the saturated model (as many parameterestimates as unique cells in the data). The next model had theinteraction term removed.

A statistical test (‘likelihood ratio test’) was carried out to see ifthere was a statistically significant difference between the models.There was no significant difference between the models (i.e. theinteraction term was not significant), so the second model was adopted(degrees of freedom in brackets):

-   -   Log of the odds of survival=intercept(1)+arm(1)++bodyweight(2)

Parameter estimates for this model are: Standard Chi- Pr > Parameter DFEstimate Error Square ChiSq Intercept 1 0.3624 0.1722 4.4267 0.0354 ARMhomeop 1 0.4167 0.1673 6.2030 0.0128 Body LO 1 −0.3009 0.2254 1.78160.1820 Weight Body MED 1 0.0267 0.2402 0.0124 0.9114 Weight

Under the null hypothesis (parameter=0) the square of ratio of theparameter estimate to its standard error has a Chi-Square distribution(1 degree of freedom). In the above table the ‘ARM (homeop)’ factor hasa P-values less than 0.05, thus we reject the null hypothesis of zeroparameter value. Odds Ratio Estimates 95% Wald Effect Point EstimateConfidence Limits ARM homeop vs placebo 2.301 1.194 4.434 BodyWeight LOvs HI 0.563 0.243 1.305 BodyWeight MED vs HI 0.781 0.322 1.896

As the estimate of ‘ARM (homeop)’ is significantly different from zero,its odds ratio has a 95% confidence interval that does not contain 1.

Discussion

Statistical analysis was the same as previously performed for theExamples 2 and 3. The end point for statistical analysis was thedifference in survival between the placebo and treatment mice. The oddsratio for survival of treatment mice relative to placebo mice was 2.301.The analysis was adjusted for mouse weight using a logistic regression(LR) model. The LR treatment odds ratio for survival of treatment micerelative to placebo mice was 2.301 and the LR treatment Chi-Square was6.2030 (1 degree of freedom), which has a P-value of 0.0128. In thisstudy we confirm that toxicity of (−)-U50488 can be counteracted byadministration of an attenuation, and in this case a potentisedpreparation of its enantiomer.

Therefore the study confirms the hypothesised method whereby thetoxicity of an optically active molecule may be decreased byadministration of its enantiomer. Although it was prospectively hoped tofirst of all determine the LD50 and then subsequently do the experimentusing 500 mice, this was not possible because there was only sufficient(−)-U50488 for 210 mice. Of these the first 52 mice were used toestablish an approximate LD50 of 25 mg/kg. Inclusion of the first 52mice transgressed the assumptions of the subsequent prospectivelyselected logistic regression model and these were excluded from theanalysis. One mouse in the placebo group was excluded because it diedimmediately after i.p. injection of (−)-U50488.

EXAMPLE 5

(+)-ephedrine hydrochloride and (−)-ephedrine hydrochloride are utilisedto modulate the locomotor activity. One isomer could be counteracted byadministration of a potentised solution of its enantiomer. This is doneby adapting the methodology described by Walker, R. B., Fitz, D.Williams, L. M. The Effects of Ephedrine Isomers and their Oxazolidineson locomoter activity in rats. Gen. Pharmac. 1993, 24:669-673. Isomersof amphetamine are also utilised.

EXAMPLE 6 Inhibition of (s)-(−)-Bay K8 644 and (S-(−)-α-MethylbenzylIsocyanate and Other Isomers with Their Respective Enantiomers or SaltsThereof in Aspergillus Awamori

Material and Methods

The fungal tests were carried out using Aspergillus awamori transformedwith the aequorin gene. Aequorin is a Ca²⁺ sensitive photoprotein, whichemits light in the dose dependent manner when bound to free [Ca²⁺].Higher luminescence means higher concentration of intracellular calcium.Higher intracellular calcium shows that the cell is perturbed. Thus, thehigher luminescence, the higher the toxicity of the compound.

Aspergillus cultures were grown on Vogel's medium in the presence of 1%sucrose in 96 well plates (E G & G Berthold, Bad Wildbad, Germany).Twelve ml of sterile Vogel's media with 1% sucrose was inoculated with1×10⁵ spores per ml. Coelenterazine was added in methanol (MeOH) to afinal concentration of 2.5 μM. The final MeOH concentration was not morethan 0.1%, which was found not to affect spore germination or hyphalgrowth (Nelson 1999). Using a 12-channel pipette (Anachem, Luton, UK),100 ml of the inoculated media was added to each well, and the platecovered with a microplate lid (Labsystems, Helsinki, Finland). Cultureswere incubated in a humidity chamber in the presence of free water at 30C for 24 h.

Luminometry was performed using an E G & G Berthold (Bad Wildbad,Germany) LB96P MicroLumat luminometer which was controlled by adedicated PC. The luminometer measures light emission in relative lightsunits (RLU). To convert RLU into [Ca²⁺]_(c) concentrations the followingempirically derived equation was used:pCa=0.332588(−log K)+5.5593,where k=luminescence counts s⁻¹/total luminescence counts (Fricker etal. 1999).

The total amount of luminescence was measured as an integral of allluminescence up to complete aequorin discharge. Aequorin was dischargedby adding 2 M CaCl₂ in 10% ethanol to the wells followed by theinjection of 100 mM CaCl₂.

4 pairs of optical enantiomers or salts thereof of the followingchemicals were used in this study:

-   -   (R)-(+)-α-Methylbenzyl isocyanate and (S)-(−)-α-Methylbenzyl        isocyanate. Only freshly made stocks of α-Methylbenzyl        isocyanide are effective. In the aqueous solution α-Methylbenzyl        isocyanate will react with water producing α-Methyl benzyl amine        and CO₂ and hence becomes a lot less toxic.    -   (R)-(+)-BAY K8644 and (S)-(−)-BAY K8644    -   (R)-(+)-nicotine (+)-di-p-toluoyltartrate salt and        (S)-(−)-nicotine    -   (R)-(+)-verapamil and (S)-(−)-verapamil

Also a racemic mixture of (R)-(+)α-Methylbenzyl isocyanate and(S)-(−)α-Methylbenzyl isocyanate was tested.

The first substance tested was BAY K8644. The effect of preincubation ofAspergillus awamori with 10 μM (R)-(+)-BAY K8644 on Ca²⁺ response to 10μM (S)-(−)-BAY K8644 is shown below in FIG. 1A.

BAY K8644 was used as a simple 10 μM dilution. 10 μM (R)-(+)-BAY K8644was used to test if there was any evidence of inhibition of the toxiceffects of its enantiomer (S)-(−)-BAY K8644.

Homeopathic preparation of all compounds were as follows:

Four compounds were prepared to investigate their ability to aggravateor inhibit toxicity in certain situations. All were stored undertemperature conditions recommended by Sigma Aldrich, who was thesupplier.

-   -   A potency of (R)-(+)-α-Methylbenzyl isocyanate was prepared to        investigate the effect on toxicity of its enantiomer,        (S)-(−)-α-Methylbenzyl isocyanate. Both of these compounds are        available as liquids at temperatures at which we used them,        i.e., greater than 3 degrees Celsius.    -   A preparation of potencies of racemic α-Methylbenzyl isocyanate        was prepared to investigate the effect on toxicity of the        racemic α-Methylbenzyl isocyanate in toxic dose. The racemic        molecule is liquid at the temperature at which we used them.    -   A potency of (R)-(+)-BAY K8644 was prepared to investigate the        effect on toxicity of its enantiomer. To do this a solution of        2.8 mM (2.8 milli Molar) (R)-(+)-BAY K8644 in distilled water        was produced, and the potency was prepared from this, as        described below.    -   A potency of (R)-(+)-Nicotine (+)-di-p-toluoyltartrate was        produced to investigate the effect on toxicity of        (S)-(−)-Nicotine. Both these compounds exist in liquid form at        temperatures used by us.    -   A potency of (R)-(+)-Verapamil hydrochloride was prepared to        investigate the effect on toxicity of its enantiomer. A solution        of 10 mM (10 milli Molar) (R)-(+)-Verapamil hydrochloride in        distilled water was produced and the potency was prepared from        this as described below.

Thirty 10 ml falcon test tubes were used to prepare the treatment andwere numbered from 1 to 30. The first test tube contained 3.7 ml 35%ethanol. The remaining 29 tubes each contained 4.7 ml 35% ethanol.

All treatment compounds were prepared as per the following example with(R)-(+)-α-Methylbenzyl isocyanate starting with, 0.2 ml of respectiveliquid compounds, or 0.2 ml of the milli Molar concentrations quoted for(+) BAY K8644 and (+) Verapamil HCl quoted above.

On the first day 0.2 ml (R)-(+)-α-Methylbenzyl isocyanate was added to3.7 ml 35% ethanol in a 10 ml Falcon test tube with cap. The test tubewas sealed and was given 20 forceful downward successions at 1-2 Hz. Onedrop was removed from this test tube using a Hirschman Laborgeräte 75 mm75 microlitre capacity non-heparinised hematocrit capillary tube, andwas added to the second test tube. This was given 20 succussions asalready described. One drop of the second test tube was removed andadded to the third test tube and so on to produce the thirtieth potency.Each level of attenuation or potency preparation was given 20succussions at 1-2 Hz, to produce until the 30^(th) potency wasprepared.

Since 35% ethanol is toxic to fungi and would have confounded theexperiment, to prepare the 30^(th) potency for use in this experiment,one drop of tube 29 was added to 4.7 ml distilled water. This wasadministered 20 succussions as previously described to produce athirtieth potency in distilled water. This is the remedy H1 which wasadministered to counteract toxicity of the (−)-isomer in one part of theexperiment. Since the stability of homeopathic preparations in water isprobably poor, it is important to administer these fresh solutions inwater to organisms within 30-40 minutes of their preparation.

Although the thirtieth potency of a propranolol isomer has previouslybeen reported as being effective in counteracting the toxic effects ofits enantiomer in mice (examples 2 and 3) it as not known if thispotency would be suitable in Aspergillus. Usually in homeopathy theelection of potency for a condition is largely determined by apractitioner's practical xperience, tradition, anecdote, by using Kent'soctave, or by trial and error—there are no validated scientificguidelines for selection of potency. Therefore to protect against anegative result on these grounds, a second preparation of homeopathy wasmade called H2.

To prepare H2 the contents of test tubes 4, 12 and 30 (all prepared in35% ethanol) were added to a 100 ml glass flask with ground glassstopper. The contents were given 20 forceful downward successions at 1-2Hz. One drop of the contents were removed using a hematocrit capillarytube and added to a 10 ml falcon test tube containing 4.7 ml distilledwater. This test tube was then succussed 20 times as already discussedto produce H2. As with H1, this H2 potency in distilled water should beadministered within 30-40 minutes of its preparation.

All homeopathic preparations were added in total volume of 25 μl ofdistilled water with the 10 min interval for a total period of 30 min.The other enantiomer was applied through the 100 μl injectors at theeffective concentration determined by the range finder screening.Controls involved the addition of relevant placebo. All the experimentswere performed at least twice with 6 replicates each. A different typeof experiment was performed with (R)-(+)-BAY K8644 and (S)-(−)-BAYK8644. Cells were preincubated with a 10 μM of (R)-(+)-BAY for 5 min andthen stimulated with the same concentration of (S)-(−)-BAY K8644. Thiswas therefore a sample dilution.

For testing the racemic mixtures of (R)-(+)-α-Methylbenzyl isocyanateand (S)-(−)α-Methylbenzyl isocyanate, separate homeopathic preparationswere prepared for both (R)-(+)-α-Methylbenzyl isocyanate and(S)-(−)-α-Methylbenzyl isocyanate isomers. To prepare the medicine orpotency for this experiment 0.2 ml (R)-(+)-α-Methylbenzyl isocyanate wasadded to 3.7 ml 35% ethanol in a 10 ml Falcon test tube with cap. Thetest tube was sealed and was given 20 forceful downward successions at1-2 Hz. One drop was removed from this test tube using a HirschmanLaborgeräte 75 mm 75 microlitre capacity non-heparinized hematocritcapillary tube, and was added to the second test tube. This was given 20succussions as already described. One drop of the second test tube wasremoved and added to the third test tube and so on to produce thethirtieth potency. Each level of attenuation or potency preparation wasgiven 20 succussions at 1-2 Hz, and this process was repeated until the30th potency was prepared. The same procedure was repeated with the (−)isomer.

One drop of 29th potency of (+) isomer was added to 4.7 ml of water.Then one drop of 29th potency of (−) isomer was added to the same tube.This was given 20 succussions as already described. This is the remedyH1. For H2 remedy one drop of 4th, 12th and 30th potencies of both (−)and (+) isomers were mixed. This was given 20 succussions as alreadydescribed. The incubation with homeopathic preparations was performed asdescribed above. The follow up stimulation was carried out using with 50mg/l mixture of (R)-(+)α-Methylbenzyl isocyanate and(S)-(−)-α-Methylbenzyl isocyanate.

25 μl of all homeopathic treatment preparations were added to eachculture well or replicate at 30 minutes, 20 minutes and 10 minutes priorto addition of the of the toxic enantiomer. The toxic enantiomer wasapplied through the 100 μl injectors at the effective concentrationdetermined by the range finder screening. Controls involved the additionof relevant placebo, which consisted of 1 drop of 35% ethanol added to4.7 ml distilled water in a 10 ml falcon test tube, and this was given20 succussions at 1-2 Hertz as previously discussed. All the experimentswere performed at least twice with 6 replicates each. The different typeof experiments was performed with (R)-(+)-BAY K8644 and (S)-(−)-BAYK8644. Cells were pre-incubated with a 10 μM of (R)-(+)-BAY for 5minutes and then stimulated with the same concentration of (S)-(−)-BAYK8644.

Results

The first experiment involved the BAY molecule and results are in FIG.1A. It was noted that final resting level was lower in the Aspergilluspreincubated with 10 μM (R)-(+)-BAY K8644. Since this microbial bioassayis able to measure more than one end-point, the final resting level wasselected as the prospective end-point for subsequent experiments howeverother end points reported here were indicative of decreased toxicity inthe treatment group.

FIG. 1A shows the effect of preincubation of Aspergillus with 10 μM(R)-(+)-BAY K8644 on Ca²⁺ response to 10 μM (S)-(−)-BAY K8644. There wassignificant inhibition of the [Ca²⁺]_(c) response and therefore therewas inhibition of toxicity. The underlined data represent statisticallysignificant data according to the 5% LSD (least significant difference)where p=0.05, n=6.

Results presented in FIG. 1B show that preincubation with 10 μM(R)-(+)-BAY K8644 caused a significant inhibition of the [Ca²⁺]_(c)response and therefore caused inhibition of toxicity.

Note that the first experiment with BAY shown in FIG. 1A and thesubsequent experiments with α-Methylbenzyl isocyanate enantiomers,except perhaps the racemic methyl benzyl experiment were performed withstrictly fresh (less than 40 minutes old) preparations in distilledwater. In fact the dely before use was between 3-5 hours. Since thestability of homeopathy in water is doubtful, it is possible that theexperiments which follow below, although significant, have demonstrateda diminished effect than would otherwise have been possible.

FIG. 1B shows the effect of homeopathic preparations of (R)-(+)-BAYK8644 on [Ca²⁺]_(c) response to 30 μM (S)-(−)-BAY K8644. H1 and H2 arehomeopathic preparations. Underlined data represents statisticallysignificant data according to the 5% LSD (least significant difference)where p=0.5, n=6. Data presented in FIG. 1B shows that homeopathicpreparations of (R)-(+)-BAY K8644 causes an increase in the [Ca2⁺]_(c)response to 30 μM (S)-(−) BAY K8644. The increase in final resting leveland recovery rate was observed. Therefore this demonstrates aggravationof toxicity.

FIGS. 2A, 2B, 2C show the effect of homeopathic preparations of(R)-(+)α-Methylbenzyl isocyanide on [Ca²⁺]_(c) response to 50 mg/l(S)-(−)α-Methylbenzyl isocyanate. Note: H1, H2 and H3 homeopathicpreparations. Underlined data represent statistically significant dataaccording to the 5% LSD (least significant difference) where p=0.05.FIGS. 2A, 2B and 2C represent repeats of the experiment performed ondifferent days.

The results presented in FIGS. 2A, 2B, 2C show that H1 has the mostvariability in its effect on [Ca2⁺]_(c) response to the 50 mg/l(S)-(−)α-Methylbenzyl isocyanate. H1 does not cause the inhibition ofthe [Ca2⁺]_(c) response and in one case causes the stimulation of[Ca2⁺]_(c) response. The variability of effect indicates that it may bepossible for homeopathic preparations to increase as well as decreasetoxicity, or in other words, to aggravate as well as ameliorate. This iswell accepted in homeopathic theory.

The amount of variability obtained with the H2 type of preparation ismuch smaller. In all cases there is a significant decrease in the[Ca2⁺]_(c) final resting level. In most cases the recovery time of[Ca2^(+]) _(c) is also significantly decreased. The effect on theamplitude is minimal and is only caused by the one-day-old H2. ThereforeH2 was selected as the primary treatment being tested in the subsequent2 experiments involving Methylbenzyl isocyanate.

Results in FIGS. 2A, 2B and 2C show that using final resting level asthe prospective end-point, the toxicity of (S)-(−)α-Methylbenzylisocyanate was inhibited with p<0.05 in each of 3 consecutiveexperiments according to the 5% least significant difference.Furthermore, the other 2 end-points reported showed trends in favour ofH2 having an inhibitory effect in toxicity, and furthermore, in over 50%of cases the reported differences were statistically significant.

The results presented in FIG. 3 shows that potencies of a racemicmixture of (R)-(+)α-Methylbenzyl isocyanate and (S)-(−)α-Methylbenzylisocyanate caused significant increase in [Ca²⁺]_(c) response. Both H1and H2 increased amplitude, resting level and recovery time of the[Ca²⁺]_(c) response therefore showing an increased toxicity of theracemic mixture to the fungal organism in comparison when usinghomeopathic preparation of (R)-(+)α-Methylbenzyl isocyanate.

FIG. 4 shows the effect of homeopathic preparations of (R)-(+)-verapamilon [Ca²⁺]_(c) response to 0.33 mM (S)-(−)-Verapamil. Note: H1 and H2homeopathic preparations. Underlined data represent statisticallysignificant data according to the 5% LSD (least significant difference)where p=0.05, n=6.

Data presented in FIG. 4 shows that homeopathic preparations of(R)-(+)-Verapamil cause an inhibition of the [Ca²⁺]_(c) response to 0.33mM (S)-(−)-Verapamil. The alleviation of the toxicity was observed indecrease in amplitude and recovery time.

FIG. 5 shows the effect of homeopathic preparations of (R)-(+)-Nicotine(+)-di-p-toluoyltartrate sale on [Ca²⁺]_(c) response to 0.5%(S)-(−)-Nicotine. Note: H1 and H2 homeopathic preparations. Underlineddata represent statistically significant data according to the 5% LSD(least significant difference) where p=0.05, n=6.

Data presented in FIG. 5 shows that homeopathic preparations of(R)-(+)-Nicotine (+)-di-p-toluoyltartrate salt causes a stimulation in[Ca²⁺]_(c) response to 0.5% (S)-(−)-Nicotine. The aggravation of thetoxicity was observed in increase in amplitude and recovery time.

EXAMPLE 7 The Effect of Enantiomer Preparations on the Toxicity ofChemicals Using a Prokaryotic Test System

Toxicity testing of various enantiomers mixtures can be done usingspecially designed lux-marked bacteria (multi-copy plasmid in which agene promoter was fused with the Vibrio fischeri luxCDABE genes). Thesebacteria used are able to detect both genotoxicity and cytotoxicity inchemicals by either increased (for genotoxicity) or reduced (forcytotoxicity) light output compared to controls without chemicals. Anychemical substance that does not elicit a response will mirror lightoutput of the control.

The bacteria are grown overnight with shaking at 27° C. in LB(Luria-Bertani) broth in the presence of antibiotic to a particular celldensity. The culture is diluted with fresh sterile LB broth withoutantibiotic and then cultured for a further 3 hour period.

Toxicity testing is done with different concentrations of chemicals in96-well microtiter plates in the presence of the bacterial test strainto determine inhibitory (cytotoxic) concentrations. A concentrationgiving inhibitory responses but not lethality is chosen for each active(−) enantiomer of the chemical for the toxicity alleviation tests.

Toxicity alleviation determination is carried out by preincubating thebacteria for 3 hours in different dilutions of the (+)-enantomer of thechemical. An overnight culture of the bacteria grown in the presence onantibiotic was diluted with fresh LB broth without antibiotic andaliquots (1 ml in a final volume of 100 ml culture) of the +enantiomerat dilutions C30, mixtures of 3 intermediate dilutions (as describedelsewhere) and a control (placebo). This 1 ml addition was repeated attimes 1 and 2 hour, and, after a 3 hour incubation aliquots of culturewere placed in microtiter plates containing the (−) chemicalconcentration which previously gave inhibitory effects. Testing overover a period of time would reveal whether the (+) enantiomer reducedthe inhibitory effect of the (−) enantiomer.

Light measurements can be made using an Anthos Lucy Luminometer. Theexperiment may also be carried out using salmonella species.

Chemicals which can be used for this experiment include enantiomers ofBAY K8644, Verapamil HCl and alpha-Methylbenzyl isothiocyanate oralpha-Methylbenzyl isocyanate or O-Acetylmandelic acid, Ibuprofen ornicotine or any optical isomer which is toxic to the microorganism usedin the bioassay which is soluble in the culture media.

EXAMPLE 8

The method of the present invention is used to counteract or enhance theeffects of any optically active molecules including protein (seeReferences 1 to 4). Toxicological investigations are used to demonstratethe method (see Reference 5).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within itsspirit and scope. The invention also includes all of the steps, featuresand compositions referred to or indicated in this specification,individually or collectively, and in any and all combinations of two ormore of said steps, features and compositions physiologically activecompound.

REFERENCES

-   1. Sempach Pty Ltd, Australian Patent—regarding drugs, proteins and    other optically active molecules, 17^(th) Sep., 2002, The Age    newspaper, Melbourne, Australia, The Age Company Limited-   2. Sempach Pty Ltd, Australian Patent—regarding drugs, proteins and    other optically active molecules, 18^(th) Sep., 2002, The    Australian, Sydney, Australia, News Limited-   3. Sempach Pty Ltd, Australian Patent—regarding drugs, proteins and    other optically active molecules, 24^(th) Sep., 2002, The Australian    Financial Review, Sydney, John Fairfax Publications Pty Ltd-   4. Sempach Pty Ltd, Australian Patent—regarding drugs, proteins and    other optically active molecules, 1^(st) Oct., 2002, The Age    newspaper, Melbourne, Australia, The Age Company Limited-   5. Sempach Pty Ltd, Advertisement in Human and Experimental    Toxicology, Vol. 21, Number 11, Nov. 2002-   6. Lovell D P, Variation in Pentobarbitone sleeping time in mice,    Laboratory Animals 1986;20:85-96 & 307-312-   7. Special Diets Services Ltd, PO Box 705, Witham, Essex, CM8 3AD,    U.K.-   8. Blackie Foundation Trust, Blackie Foundation Trust Protocol,    London, U.K.-   9. Walker R B, Fitz D, Williams L M, The effect of ephedrine isomers    and their oxazolidines on locomotor activity in rats, Gen Pharmac.    1993;24:669-73-   10. Hennekens, C. H. and Buring, J. E., Epidemiology in Medicine,    258-264, 1987, Philadelphia, Lippincott Williams & Wilkins-   11. Hamilton Laboratory Glass Ltd, Europa House, Margate, Keng,    England, U.K. Unit 1 Westwood Industrial Estate, Ramsgate Road-   12. Kuzeff, R. M., Mecheva, R. P., and Topashka-Anchera, M. N.,    Inhibition of (−)-Propranolol hydrochloride by its enantiomer in    white mice (2002) (Unpublished)-   13. Kuzeff, R. M., Mecheva, R. P., and Topashka-Anchera, M. N.,    Inhibition of (−)-Propranolol hydrochloride by its enantiomer in    white mice (2) (2002).-   14. Nelson, G. 1999. Calcium measurement using recombidant aequorin.    PhD Thesis Edinburgh University.

1. (canceled)
 2. A method of treating an organism suffering from theeffects of a chemical agent, said method being selected from: (i)administering a dilution or an ultra-high dilution or potentisedpreparation of a stereoisomer of said chemical agent; (ii) potentising astereoisomer of said chemical agent and administering said potentisedstereoisomer to the organism; (iii) diluting a stereioisomer of saidchemical agent and administering said diluted stereoisomer to theorganism; and (iv) diluting a stereoisomer of said chemical agent to anultra-high dilution of a stereoisomer of said chemical agent andadministering said ultra-high diluted stereoisomer to the organism. 3.(canceled)
 4. (canceled)
 5. The method according to claim 2, whereinsaid stereoisomer is selected from the group consisting of opticalisomers and geometric isomers.
 6. The method according to claim 5,wherein said optical isomers comprise one or more diastereoisomers. 7.The method according to claim 5, wherein said optical isomers compriseone or more enantiomers.
 8. The method according to claim 7, whereinsaid stereoisomer is the enantiomer of the most active stereoiosomer ofsaid chemical agent.
 9. The method according to claim 2, wherein saidstereoisomer is the least active stereoisomer of said chemical agent.10. The method according to claim 2, wherein said chemical agent is aphysiologically active compound.
 11. The method according to claim 2,wherein said chemical agent is selected from the group consisting ofpharmaceuticals and drugs of addiction.
 12. The method according toclaim 2, wherein said chemical agent is propranolol.
 13. The methodaccording to claim 2, wherein said chemical agent is selected from thegroup consisting of opioid receptor agonists, barbiturates, andamphetamines.
 14. The method according to claim 2, wherein said chemicalagent is selected from the group consisting of morphine, nicotine, andheroin.
 15. The method according to claim 2, wherein said chemical agentis a naturally occurring compound is selected from the group consistingof snake venom and adrenaline.
 16. The method according to claim 2,wherein said chemical agent is a stereoisomer which undergoes or hasundergone in vivo transformation into a physiologically active form. 17.The method according to claim 2, wherein the chemical agent is selectedfrom the group consisting of angiotensin 2 receptor antagonists,angiotensin converting enzyme inhibitors, and adrenergic agonists. 18.(canceled)
 19. (canceled)
 20. The method according to claim 2, whereinsaid chemical agent is selected from the group consisting of enalapril,enalaprilat, atacand, candesartan, dipivefrine hydrochloride, andadrenaline.
 21. The method according to claim 2, wherein said chemicalagent is a calcium channel agonist.
 22. The method according to claim 2,wherein said chemical agent is a physiologically active compoundselected from the group consisting of BAY K8644, Verapamil HCL,α-methylbenzyl isocyanate, trans-U-50488, and ephedrine.
 23. (canceled)24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled) 28.(canceled)
 29. The method according to claim 7, wherein said chemicalagent is (S)-(−)-Propranolol and said enantiomer is (R)-(+)-Propranolol.30. The method according to claim 7, wherein said chemical agent is(R)-(+)-Propranolol and said enantiomer is (S)-(−)-Propanolol.
 31. Themethod according to claim 7, wherein said chemical agent is(−)-trans-(1S,2S)-U-50488 and said enantiomer is(+)-trans-(1R,2R)-U-50488.
 32. The method according to claim 7, whereinsaid chemical agent is (+)-trans-(1R,2R)-U-50488 and said enantiomer is(−)-trans-(1S,2S)-U-50488.
 33. The method according to claim 7, whereinsaid chemical agent is (S)-(−)-BAY K8644 and said enantiomer is(R)-(+)-BAY K8644.
 34. The method according to claim 7, wherein thechemical agent is (R)-(+)-BAY K8644 and said enantiomer is (S)-(−)-BAYK8644.
 35. The method according to claim 7, wherein said chemical agentis (S)-(−)-α-methylbenzyl isocyanate and said enantiomer is(R)-(+)-α-methylbenzyl isocyanate.
 36. The method according to claim 7,wherein said chemical agent is (R)-(+)-α-methylbenzyl isocyanate andsaid enantiomer is (S)-(−)-α-methylbenzyl isocyanate.
 37. The methodaccording to claim 2, wherein said stereoisomer is potentised bysuccession, agitation, and/or trituration,
 38. The method according toclaim 2, wherein the attenuation of said stereoisomer is selected fromthe group consisting of 4^(th), 6^(th), 12^(th), 15^(th), 30^(th),200^(th), and 1000^(th) attenuation.
 39. The method according to claim2, wherein said stereoisomer comprises a dilution or potency orattenuation existing below the toxic range of said chemical agent andproduces a stimulatory or inhibitory effect.
 40. The method according toclaim 2, wherein said stereoisomer comprises different dilutions orpotencies or attenuations added together.
 41. The method according toclaim 7, wherein said chemical agent comprises a racemic mixturecontaining both (+)- and (−)-enantiomers of a stereoisomer of saidchemical agent and a mixture of one or more dilutions or potencies orattenuations of each enantiomer is administered.
 42. The methodaccording to claim 41, wherein said mixture of one or more dilutions orpotencies comprises a mixture of equal or unequal volume of one or moresame or different dilutions or potencies or attenuations of said eachenantiomer.
 43. The method according to claim 42, wherein said mixturecomprises the 4^(th), 12^(th) and 30^(th) dilutions or potencies orattenuations of one enantiomer added to the 4^(th), 12^(th) and 30^(th)dilutions or potencies or attenuations of the opposite enantiomer. 44.The method according to claim 2, wherein said chemical agent is ionicand said stereoisomer is derived from an ionic form of said chemicalagent.
 45. The method according to claim 2, wherein said chemical agentis a salt and said stereoisomer is derived from a salt of said chemicalagent.
 46. The method according to claim 2, wherein said chemical agentis ionic or a salt and said stereoisomer is derived from a non-ionic ornon-salt form of said chemical agent.
 47. The method according to claim2, wherein said chemical agent is non-ionic or in a non-salt form andsaid stereoisomer is derived from a ionic form or salt of said chemicalagent.
 48. The method according to claim 2, wherein the organism isselected from the group consisting of micro-organisms, animals, plants,and humans.
 49. The method according to claim 48 wherein saidmicro-organism is a fungus.
 50. The method according to claim 49 whereinsaid fungus is Aspergillus awamori.
 51. The method according to claim 48wherein said micro-organism is a bacteria.
 52. The method according toclaim 51 wherein said bacteria is Vibrio fisheri.
 53. The methodaccording to claim 2, wherein the organism is an animal.
 54. The methodaccording to claim 2, wherein the organism is a mouse.
 55. The methodaccording to claim 2, wherein the organism is a human.
 56. The methodaccording to claim 55, wherein said stereoisomer is administered orally,sublingually, intravenously, transdermally, subcutaneously,intraperitoneally, or via a mucous membrane.
 57. (canceled) 58.(canceled)
 59. The method according to claim 55, wherein saidstereoisomer is administered by a bioresonance or electrodermal testingdevice.
 60. The method according to claim 59, wherein said bioresonanceor electrodermal testing device is selected from the group consisting ofa MORA machine, Listen Machines and a Vega Select Machine. 61.(canceled)
 62. (canceled)
 63. (canceled)
 64. (canceled)
 65. (canceled)66. (canceled)
 67. (canceled)
 68. (canceled)
 69. (canceled) 70.(canceled)
 71. (canceled)
 72. (canceled)
 73. (canceled)
 74. (canceled)75. (canceled)
 76. (canceled)
 77. (canceled)
 78. (canceled) 79.(canceled)
 80. (canceled)
 81. (canceled)
 82. (canceled)
 83. (canceled)84. (canceled)
 85. (canceled)
 86. (canceled)
 87. (canceled) 88.(canceled)
 89. (canceled)
 90. (canceled)
 91. (canceled)
 92. (canceled)93. (canceled)
 94. (canceled)
 95. (canceled)
 96. (canceled) 97.(canceled)
 98. (canceled)
 99. (canceled)
 100. (canceled)
 101. (canceled)102. (canceled)
 103. (canceled)
 104. (canceled)
 105. (canceled) 106.(canceled)
 107. (canceled)
 108. (canceled)
 109. (canceled) 110.(canceled)
 111. (canceled)
 112. (canceled)
 113. (canceled) 114.(canceled)
 115. The method according to claim 53, wherein saidstereoisomer is administered orally, sublingually, intravenously,transdermally, subcutaneously, intraperitoneally, or via a mucousmembrane.
 116. The method according to claim 53, wherein saidstereoisomer is administered by a bioresonance or electrodermal testingdevice.
 117. The method according to claim 116, wherein saidbioresonance or electrodermal testing device is selected from the groupconsisting of a MORA machine, Listen Machine, and a Vega Select Machine.118. The method according to claim 2, wherein the potency of saidstereoisomer is selected from the group consisting of 4^(th), 6^(th),12^(th), 15^(th), 30^(th), 200^(th), and 1000^(th) potency.
 119. Themethod according to claim 2, wherein the dilution of said stereoisomeris selected from the group consisting of 4^(th), 6^(th), 12^(th),15^(th), 30^(th), 200^(th), and 1000^(th) dilution.
 120. The methodaccording to claim 2, wherein said chemical agent is a beta receptorblocker.
 121. A method of treating an organism suffering from theeffects of a chemical agent, which agent is characterized by one or morechiral centers, said method being selected from: (i) administering adilution or an ultra-high dilution or potentised preparation of astereoisomer of said chemical agent; (ii) potentising a stereoisomer ofsaid chemical agent and administering said potentised stereoisomer tothe organism; (iii) diluting a stereioisomer of said chemical agent andadministering said diluted stereoisomer to the organism; and (iv)diluting a stereoisomer of said chemical agent to an ultra-high dilutionof a stereoisomer of said chemical agent and administering saidultra-high diluted stereoisomer to the organism.